Many inherited connective tissue diseases result from a point mutation or frame-shift mutation. A gene therapy capable of correcting such mutations would be an advance in treating severe and nondurable diseases such as hypophosphatasia, chondrodysplasia and perhaps even osteogenesis imperfecta. Current protocols rely on the expression of a normal gene under the control of a heterologous promoter in cells that harbor a defective copy of the gene. One obstacle in such treatments is the failure of the transferred gene to recapitulate faithfully the expression pattern of the normal endogenous gene. Thus, gene therapy, capable of correcting such the mutation at the genetic level is preferable. The success rate of this approach however in mammalian cells has been hampered by an extremely low frequency of targeting and interference from the illegitimate recombination pathway that takes place independent of DNA sequence homology. We developed an experimental strategy that centers around site-specific correction of mutations using a unique chimeric oligonucleotide containing both RNA and DNA and the enzymatic activity of a eukaryotic recombinase. These oligonucleotides increase the specificity of homologous alignment by the recombinase and facilitate the recognition and correction of the mismatched base on the genomic strand by the endogenous DNA repair system. We have also cloned and over-expressed a eukaryotic analog of the RecA protein from Ustilago maydis, designated as Rec2. This protein catalyzes homologous pairing of a wide variety of DNA substrates and promotes homologous alignment of the chimeric molecule with its genomic target. Thus, used in conjunction with chimeric oligonucleotides, the Rec2 protein can provide a highly specific and regulatable gene targeting system. In our preliminary studies, we demonstrated that a chimeric oligonucleotide designed to correct a point mutation (G to A) at position 711 of the human bone/liver/kidney alkaline phosphatase cDNA converted an enzymatically inactive alkaline phosphatase cDNA to wild type form. A high frequency of a correction event was observed at low concentration of oligonucleotide of specific sequence. Furthermore, we demonstrated that the chimeric oligonucleotides were extremely stable in both serum and inside cells. The high efficacy, specificity and stability of chimeric oligonucleotides make them an attractive candidate for gene therapy utilizing nucleic-acid-based therapeutics. This particular mutation at position 711 has been identified in patients with the prenatal form of hypophosphatasia, which results in defective skeletal mineralization. In this proposal, we will extend our preliminary results to more clinically relevant protocols by attempting to correct mutations of genomic sequences involved in several inherited connective tissue diseases.